JPH0995885A - Bleaching of pulp - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for bleaching pulp, capable of bleaching the pulp in non-polluting state perfectly not using chlorine by combining an oxygen- bleaching method with a lignin peroxidase treatment method. SOLUTION: This method for bleaching the pulp comprises suspending oxygen-bleached pulp in an amount of 0.1-30wt.%, preferably 0.5-10wt.%, in water, adding lignin peroxidase having an activity of 0.0001-1.0U/ml without adding a substituted aromatic compound such as veratryl alcohol and subsequently bleaching the pulp in a pH of 2-7, preferably 3-5, at a temperature of 20-50 deg.C, preferably 30-40 deg.C, for 30min to 48hr, preferably 1-20hr.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for bleaching a pulp using an enzyme, and particularly to a method for bleaching oxygen-bleached pulp with 0.00
It relates to a method for bleaching pulp, which is treated with lignin peroxidase having an activity of 01 to 1.0 U / ml.

[0002]

2. Description of the Related Art Conventionally, a large amount of coloring substances, mainly lignin, remains in pulp after cooking in the bleaching process of pulp. Therefore, a bleaching step is required to remove the coloring substances from the pulp after cooking. However, since chlorine, chlorine dioxide, or chlorine-based chemicals such as sodium hypochlorite are used in large amounts in this bleaching step, chlorine that significantly corrodes metals remains in the waste liquid, and during the digestion, It is difficult to use the concentrated combustion in the chemical recovery process and the recycle use in the bleaching process.

Further, recently, it has been revealed that the effluent discharged from the system after treating the bleaching waste liquid with activated sludge or coagulating sediment contains chlorinated phenols such as dioxins, which are extremely toxic. It became clear that there is a possibility of causing pollution to the natural environment. Therefore, an oxygen-based bleaching method such as oxygen / alkali treatment which does not use any chlorine-based bleaching agent, and a bleaching method called biobleaching using microorganisms and enzymes have attracted much attention.

In the oxygen / alkali bleaching treatment, under a normal alkaline condition, an acidic hydroxyl group such as a phenolic hydroxyl group in lignin is liberated to form a highly charged portion, and oxygen is electrophilically reacted therewith. This is a method of decomposing lignin, which is a bleaching method that can be performed relatively easily because the reaction proceeds by simply blowing compressed air under alkaline conditions, and is widely adopted. However, in this bleaching method, because of its reaction mechanism, only the portion having a free phenolic hydroxyl group in the lignin can be decomposed, and therefore, in practice, only about half of the lignin in the pulp after cooking can be removed. Can not.

On the other hand, in the biobleaching bleaching method, selective bleaching can be carried out on the basis of the substrate specificity of the enzyme, and further bleaching can be carried out at room temperature, atmospheric pressure and neutral range. It has the advantage that no special performance is required and the danger of the chemicals used is extremely low. Also, when using the biobleaching bleaching method,
Examples of microorganisms having an enzyme capable of decomposing lignin, which is a coloring substance in pulp, include a series of lignin degrading microorganisms called white rot fungi.

As a report of pulp bleaching using such microorganisms, an example using Phanerochaete chrysosporium (Biotech Letter. I, 347 to 353 (1979)), and Cori
Example using olisversicolor (Tappy Journal. 198
May 1997 issue 217-220). However, the biobleaching treatment with such microorganisms has a drawback that the treatment time is long and that the lignin is decomposed and at the same time the cellulose is decomposed.

Therefore, in order to shorten the processing time, it is considered that the enzyme produced by these microorganisms is used for direct bleaching. Conventionally, many attempts have been made to decompose lignin with an enzyme and use it for bleaching in the pulp and paper industry, and for this purpose, lignin peroxidase, manganese peroxidase, laccase, and other white-rot fungi are produced. Phenol oxidase is used.

Specific examples thereof include P. chrysosporiu
Using chemically modified lignin peroxidase of m,
A method of bleaching pulp and treating wastewater in the absence of hydrogen peroxide (Japanese Patent Laid-Open No. 3-104993), P. chrysospor
lignin peroxidase rL of SC26 mutant of ium
A method of pulp treatment using DM or a method of decolorizing bleaching wastewater (Japanese Patent Publication No. 62-500604) is disclosed. However, there is a drawback in that the lignin remaining in the pulp cannot be easily decomposed by bleaching with only the enzyme.

Therefore, multi-stage bleaching was carried out using the above-mentioned enzyme in combination with several kinds of chemicals. For example, after lactase treatment and bleaching treatment using chlorine dioxide or a mixture of chlorine dioxide and chlorine gas, further alkali extraction (Japanese Patent Laid-Open No. 3-130485), redox potential in the range of 200 to 500 mV. A method is disclosed in which the bleaching reaction is started by adjusting and then adding a lignin-degrading enzyme (Japanese Patent Laid-Open No. 5-505857).

In general, in bleaching with chemicals, it is necessary to select a chemical having a proper reactivity with the chemical structure of lignin in pulp. That is, this point becomes particularly important when trying to reduce chlorinated phenols which are toxic substances. However, in the invention of multi-stage bleaching described above, it is difficult to say that the lignin-degrading enzyme is effectively used because the point is not taken into consideration, and it is not possible to completely suppress the generation of chlorinated phenols. Of course, it was not possible to remove the chlorine-based residue.

In contrast to the above method, as a chlorine-free multi-stage bleaching method, the activity is 0.05 to 10.0 U / ml under a steady state concentration of hydrogen peroxide of about 0.001 to 0.5 mM.
There is also disclosed a bleaching method using lignin peroxidase or manganese peroxidase, which is
No. 80687).

By the way, when lignin peroxidase decomposes lignin, a two-step reaction occurs: an enzymatic reaction in which lignin peroxidase abstracts an electron from a substrate, lignin, and a subsequent non-enzymatic radical reaction [(K. Hammel , Proc.Natl.Acad.Sci.USA, 83,3708,198
6), (K. Hammel et al., J. Biol. Chem., 260, 8348, 1985)]. Since this second-stage reaction is a random reaction due to the generated cation radicals, the concentration of this radical is an important factor in the reaction. That is, if the concentration of radicals is high, the radicals will recombine and the reaction will proceed to polymerization rather than decomposition.

Therefore, the activity is about 0.05-10.0 U /
Even if bleaching with the above-mentioned lignin peroxidase or manganese peroxidase, which is the amount of ml, is high, lignin peroxidase and Mn peroxidase have high concentrations, so that a high decomposition rate of lignin cannot be obtained. Further, in the bleaching with the above-mentioned enzyme, there was a drawback that a coenzyme such as veratryl alcohol or manganese sulfate had to be added.

[0014]

Therefore, as a result of diligent studies on a method of utilizing lignin peroxidase, the inventors of the present invention identified oxygen bleached pulp in accordance with each chemical reaction mechanism in the process of decomposing lignin. We have found that treatment with lignin peroxidase having a range of activities is particularly effective and have completed the invention. Therefore, it is an object of the present invention to provide a pollution-free and efficient pulp bleaching method.

[0015]

The above object of the present invention is to add oxygen bleached pulp to 0.0001 to 1.0 U / ml.
It was achieved by a method of bleaching pulp, characterized in that it was treated with lignin peroxidase having the activity of. Lignin peroxidase (hereinafter abbreviated as LiP) used in the present invention refers to P. chrysosporium in 1983.
The enzyme isolated and purified from the culture fluid of M. Tien et al., Science 221,661,1983 or J. Glenn et al., Biochem.
ophys.Res.Commun., 114,1077,198), which contains heme iron in the active center, and generally veratryl alcohol (3,4-dimethoxy-phenol) is converted to veratrumaldehyde in the presence of hydrogen peroxide. It exhibits activity as it is oxidized.

As a bacterium for obtaining LiP, a bacterium capable of decomposing lignin can be generally mentioned, and a specific example thereof is Coriolus versicolor IFO 3034.
0 etc., Pleurochaete chrysosporiu
m ATCC 34541 etc.), Trametes dickins
ii IFO 6488, etc.), Tamachorei (Polyporus mikado)
i IFO 6517 etc.) and the like.

The method for isolating LiP in the present invention varies depending on the strain used, but it can be isolated by a commonly used method. Examples of this method include M.
H. Gold et al. (Arch. Biochem. Biophy., 234, 353-362, 19
84), Renganathan et al. (Arch. Biochem. Biophy., 24
1,304-314,1985), F. Tonon & E. Odier's method (Appl. En.
viron.Microbiol., 54,462-472,1988), the method of A. Jager et al. (Appl.Envrion.Microbiol., 50,1274-1288,1985), T.
Kirk et al. (Enzyme Microbiol.Technol., 8, 27, 1986)
, M. Kuwahara et al. (Mokuzai Gakkaishi, 33,821,198
7) etc. are known.

The enzyme used in the present invention does not need to be purified, and may have LiP activity. In order to suppress the concentration of radicals generated by the decomposition of lignin by LiP to a low level, the activity of LiP in the present invention is set to 0.
It is necessary to be in the range of 0001 to 1.0 U / ml, and particularly preferably in the range of 0.001 to 0.1 U / ml.

When the activity is higher than 1.0 U / ml, L
The chances of recombination of lignin radicals generated by iP increase, and the increase in whiteness due to bleaching is suppressed. On the contrary, when the activity is lower than 0.0001 U / ml, the reaction does not proceed, and the bleaching effect is not exhibited.

As the oxygen bleaching pulp used in the present invention, pulp which has been subjected to delignification by reacting with oxygen under heat and pressure under alkaline conditions can be used.
When treating the pulp in the present invention, first, oxygen bleached pulp is usually suspended in water within the range of 0.1 to 30% by weight, preferably 0.5 to 10%, and the enzyme lignin peroxidase is added to the treatment. .

When the pulp concentration is 0.1% or less, the contact between the enzyme and the pulp is insufficient and the treatment efficiency is lowered. On the other hand, when the pulp concentration is 30% by weight or more, the water content is too small and the enzyme is poorly dispersed, so that the reactivity is lowered. descend. PH of the treatment liquid at that time
The temperature and the temperature are not particularly limited as long as the enzyme is not deactivated, but the pH is usually 2 to 7, preferably 3
~ 5, the temperature is 20 to 50 ° C, preferably 30 to 4
It may be in the range of 0 ° C. The reaction time varies depending on the treatment conditions, but is usually 30 minutes to 48 hours, preferably about 1 to 24 hours.

In the present invention, lignin can be decomposed without adding an aromatic compound such as veratryl alcohol. The reason for this is not clear, but it is inferred as follows. That is, conventionally, veratryl alcohol has been said to function as a radical carrier between the enzyme and the pulp, which is a substrate, but recent findings show that the potential difference from the active site of lignin peroxidase to the surface is higher than that of a charge relay. It is presumed that the movement causes a difference in charge on the surface of the enzyme, where electrons are exchanged with the pulp and the reaction proceeds.

[0023]

DETAILED DESCRIPTION OF THE INVENTION The method of the present invention can be applied in any case where the enzyme bleached pulp is further bleached. After the pulp after cooking is simply subjected to ozone treatment or oxygen / alkali bleaching treatment, it is also possible to carry out once dried oxygen bleaching pulp, as a step after the bleaching treatment.
It can also be carried out continuously without obtaining a dry oxygen bleached pulp. In addition, it can be carried out as the final step of the conventional bleaching processing such as multi-step processing or as an intermediate step.

[0024]

INDUSTRIAL APPLICABILITY According to the present invention, the bleaching treatment with LiP and the oxygen-based bleaching treatment are effectively combined in accordance with the decomposition reaction mechanism of lignin, so that it is possible to perform bleaching with low enzyme activity. Since it is not necessary to add an aromatic compound such as tolyl alcohol, it is possible to bleach completely without chlorine and without pollution.

[0025]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.
Unless otherwise specified, “parts” and “%” described below mean “parts by weight” and “% by weight”, respectively.

Preparation of Kirk's salt solution After dissolving each reagent in pure water as shown in Table 1, the pH was adjusted to 4.5 with HCl or NaOH to prepare a Kirk's salt solution.

TABLE 1 _{────────────────── KH 2 PO 4 20g MgSO 4} · 7H 2 O 5g CaCl 2 1g mineral solution 100ml Thiamine HCl 10mg of pure water 1000 ml ──── ──────────────

Preparation of Mineral Solution in Table 1 After each reagent was dissolved in pure water as shown in Table 2, pH was adjusted to 4.5 with HCl or NaOH to prepare 1 liter of mineral solution.

TABLE 2 ─────────────────── nitrilotriacetate _{1.5g MgSO 4 · 7H 2 O 3.0g} MnSO 4 · 7H 2 O 0.5g NaCl 1.0g FeSO 4 · 7H ₂ O 100mg CoSO ₄ 100mg CaCl ₂・ 2H ₂ O 100mg ZnSO ₄・ 5H ₂ O 10mg AlK (SO ₄ ) ₂ 10mg H ₂ O ₂ 10mg NaMoO ₄ 10mg ─────────────── ────

Lignin pero using P. chrysosporium
Preparation of xydase Gold et al. (Gold et al., Arch. Biochem. Bioph
y., 234,353-362,1984) and (Renganathan, V., Miki,
K. and Gold, MH, Arch. Biochem. Biophy., 241, 304-314,
1985) was slightly modified and prepared by 4 steps of inoculum culture, preculture (liquid stationary culture), large-scale shaking culture, and filtration.

Inoculum culture A slant was prepared according to the medium composition shown in Table 3 below.
P. chrysosporium was inoculated, cultured at 30 ° C for 1 week to form spores, and stored in this state at 4 ° C. Medium composition:

[Table 3] ──────────────────────── Malt extract 3% Yeast extract 0.15% Kirk salt solution 10 m / 100 m Ammonium tartrate 24 mM Agar 1. 5% ────────────────────────

Pre-Culture (Liquid Static Culturing) After dissolving each medium composition shown in Table 4 below in pure water, the pH was adjusted to 4.5 with HCl or NaOH to prepare 120 ml of a medium solution. Medium composition:

[Table 4] ──────────────────────── Glucose 1.2g (1%) Ammonium tartrate 1.2ml (1.2mM) Kirk salt solution 12ml 2,2 -Dimethylsuccinic acid 12 ml (20 mM) ────────────────────────

The obtained culture medium solution is divided into 20 ml portions and 2
The mixture was dispensed into a 00 ml Erlenmeyer flask and sterilized by an autoclave. At the same time, 9 ml of sterilized water was added to the slant obtained in the process of culturing the bacterial seeds, the surface of the medium was scratched with a spatula, and the spores were suspended in water. The resulting spore suspension water 0.1
ml was pipetted, inoculated into a sterilized 200 ml Erlenmeyer flask medium, and statically cultured at 37 ° C. for 3 days.

Mass culture (shaking) Each medium composition shown in Table 5 below was dissolved in pure water, and the pH was adjusted to 4.5 with HCl or NaOH to prepare 1.5 liters of medium solution. Medium composition:

[Table 5] ──────────────────────── Glycerin 15g (1%) Ammonium tartrate 15ml (1.2mM) Kirk salt solution 150ml Mineral solution 90ml Sodium acetate 200ml (20mM) Twin 80 7.5g (0.5%) ─────────────────────────

The obtained culture medium solution was dispensed into a 500 ml Erlenmeyer flask (using PYREX product) in 250 ml portions, and the flask was stoppered with a silicon stopper through a glass tube for oxygen purging, and the glass tube was covered with aluminum foil. After covering with, it was put in an autoclave for 15 minutes for sterilization. Pre-incubate one cell mat flask with a Waring blender for 2
After homogenizing for about 0 seconds, 50
After inoculating into a 0 ml Erlenmeyer flask and repeating 6 times for 6 flasks, 37 ° C., 140 r.p.m. p. m. Shaking culture was performed. To induce LiP on the 3rd day of culture, 0.252 g of veratryl alcohol was added to each flask and oxygen purging was started. Oxygen purging was performed through the glass tube of the culture flask for a minimum of 1 hour per day.

(Culture liquid) As the filtered LiP is produced, the culture liquid is colored from orange to brown. Since the color of the medium began to darken, L
The activity of iP was measured, and when the high activity was obtained, the culture was stopped, the cells were immediately filtered with gauze to separate the cells, and then the filtrate was centrifuged. The supernatant was filtered with a membrane filter (pore size 0.45 μm) while further cooling. The obtained filtrate was purified using an ion exchange column, and the LiP active fraction and the manganese peroxidase active fraction were collected.

Measurement of Lignin Peroxidase Activity In order to measure the activity of the obtained lignin peroxidase, the mixture samples shown in Table 6 below were put in a 3 cc test tube, and 3
After heating by shaking in a constant temperature bath at 7 ° C for 10 minutes, 20 µl of 50 mM H ₂ O ₂ except for the control and pure water for the control were added, and the mixture was further shaken at 37 ° C for 5 minutes and then immersed in a water bath. The test tube was transferred to a stainless steel test tube stand and kept warm.

Measurement liquid composition:

[Table 6] ──────────────────────── 20 mM succinate buffer (pH 3.0) 1480 μl 10 mM veratryl alcohol buffer 100 μl lignin peroxidase (culture filtrate Etc.) 400 μl ────────────────────────

The absorbance of the obtained reagent before and after the reaction at 310 nm was measured, and the specific activity of the enzyme was calculated from the following formula.

[Equation 1] Where t is reaction time, At is absorbance after reaction, A _t
o is the absorbance before the reaction, * is 31 of veratrum aldehyde
It is a molar extinction coefficient at 0 nm.

Manganese peroxidase activity measurement Manganese peroxidase activity was determined by the method of Pais et al.
Ice et al., Appl. Environ. microbiol., 59 (1993) 260) was performed by measuring the change in absorbance at 270 nm using Mn as a substrate. 270n per minute
The amount of enzyme that changes the absorbance of m by 1 was set to 1U.

Example 1. LiP with lignin peroxidase bleaching activity of 0.08 U / ml, activity of 0.05 U / ml
ml glucose oxidase, glucose 50 mM,
A solution containing pH 4 (adjusted with 2,2-dimethylsuccinic acid) and 0.05% of a surfactant (Twin 80) was prepared, and using this, unbleached pulp (UKP) manufactured by the factory and pulp after oxygen bleaching (OKP) 4g (dry) each 3
The bleaching reaction was performed under the conditions of 7 ° C., 140 rpm, and 24 hours. After the reaction was completed, handmade paper was prepared and whiteness (JISP8
The results of measuring 123) are as shown in Table 7.

[0040]

[Table 7]

Example 2. OKP was prepared in the same manner as in Example 1 except that the activity of LiP was changed to that shown in Table 8.
The results of bleaching are shown in the table.

[Table 8] From the results shown in Table 8, it is demonstrated that the bleaching of pulp after oxygen bleaching is remarkable when the activity of LiP is 0.0001 to 1.0 U / ml, and particularly the activity of LiP is preferably in the range of 0.001 to 0.1 U / ml. Was done.

Example 3. Veratryl alcohol (VA) as a coenzyme at 0, 0.005, 0.05, 0.5, 5 mM
In the same manner as in Example 1, except that
The results of bleaching P are shown in Table 9.

[0043]

[Table 9] From the results in Table 9, it was demonstrated that the bleaching of the pulp by LiP did not depend on the coenzyme (veratryl alcohol).

Example 4. The oxygen-bleached pulp bleached with LiP in Example 1 was subjected to N. Liebergott's conditions (Tappi.
Further ozone bleaching according to J., 75 (1) 145 (1992)), the whiteness increased to 86.8%.

Comparative Example 1 LiP with a bleaching activity of 0.08 U / ml of pulp by manganese peroxidase , an activity of 0.1 U / ml
The results of bleaching the pulp with LiP in exactly the same manner as in Example 1 except that 0.5 mM of manganese sulfate was added instead of ml of manganese peroxidase were obtained.
As shown in.

[0046]

[Table 10]

From the results shown in Tables 7 and 10, in the pulp bleaching with LiP, a larger increase in whiteness was observed when the pulp after oxygen bleaching was observed, and manganese peroxidase was able to bleach UKP. However, since OKP cannot be bleached so much, it was demonstrated that LiP is suitable for enzymatic bleaching of OKP.

Comparative Example 2 The oxygen bleached pulp used in Example 1 was bleached with a chlorine-based chemical under the conditions shown in Table 11 below to obtain a pulp having a whiteness of 87.2%. Bleaching conditions with chlorine chemicals:

[Table 11]

Analysis of dioxin The amount of dioxin in the pulp prepared in Example 1 and Comparative Example 2 was analyzed according to the following procedure. A predetermined amount of air-dried pulp was packed in a Soxhlet extractor, 100 μl of toluene as an internal standard substance was added to the pulp, and extraction was performed with ethanol for 48 hours. Four drops of n-tetradecane, which is a dioxin-retaining agent, was added to this extract, and the mixture was concentrated to 1 ml at 35 ° C. under reduced pressure using a rotary evaporator. This concentrated solution was dissolved in n-hexane and applied sequentially to a sulfuric acid silica gel column, an alumina column, and an activated carbon column. Next, 10 μl of n-tetradecane was added to the eluate and air-dried in a draft to prepare a sample for analysis. The results of the high-resolution gas chromatograph rough mass spectrometry analysis of the sample thus prepared are shown in Table 12.

[0050]

[Table 12] ──────────────────────── Example 1 Comparative Example 2 ───────────────── ──────── Undetectable 1.8 ng / kg ──────────────────────── From the results of Table 12, the results of Example 1 are shown. It was demonstrated that the pulp is dioxin-free and that it is safer than regular chlorine bleached pulp.

Claims (2)

[Claims] 1. An oxygen bleached pulp is prepared from 0.0001-1.
A method for bleaching pulp, which comprises treating with lignin peroxidase having an activity of 0 U / ml. 2. The pulp bleaching method according to claim 1, wherein a substituted aromatic compound is not added.